Isolation of Listeria monocytogenes from poultry red mite (Dermanyssus gallinae) infesting a backyard chicken farm in Greece

The poultry red mite (PRM), Dermanyssus gallinae, is arguably the most harmful, ubiquitous haematophagous ectoparasite infesting egg-laying hens. PRM is a vector of various microorganisms, with some being important for food microbiology and public health. The present study aimed to investigate the presence of specific pathogens, including Escherichia coli, Salmonella spp. and Listeria spp., carried by PRM infesting a chicken farm in Greece. Mites were caught using cardboard traps (Avivet), and 100 unwashed PRM were homogenized and used for microbiological cultures. Microbiological cultures were carried out on general and selective substrates to detect the above-mentioned bacteria. Specifically for Listeria spp., DNA was extracted from bacteria grown in Tryptone Soya Yeast Extract Agar using a commercial kit. The hly gene encoding the Listeriolysin O protein was amplified by PCR. Mites were identified as D. gallinae using morphological keys as well as by COI DNA barcoding. Microbiological cultures and PCR assays were positive for Listeria monocytogenes. No other bacteria were detected. The current study constitutes the first molecular isolation of L. monocytogenes from D. gallinae, confirming that PRM can carry this food-borne pathogen. PRM control measures and hygiene practices should be applied to minimize any possible contamination risk of poultry products with L. monocytogenes and safeguard public health.

www.nature.com/scientificreports/ and adults are hematophagous. Nymphs feed to moult to the next stage. Female adults feed to lay eggs, while male adults only feed periodically. Surprisingly, female mites can survive for 9 months without feeding 7 . Usually, the PRM feeds during the night, in darkness, for approximately 1 h every 2-4 days 1,8,9 . It prefers to feed on the hen's body parts that are not covered with feathers, such as the breast and lower legs 10 , or from superficial veins on the neck and back 11 . Throughout the day, D. gallinae hides in the hens' environment, specifically cracks and crevices, under the egg belt or metal connections of cages, inside perches, or in the chickens' nests 1 . They gain access to the host by travelling through the poultry house equipment and climbing up their legs or falling from the ceiling 11 . Unfed mites have a pale grey colour and, in contrast, engorged mites have a brown to bright red colour 12 . Adult females can drink 204 μg of blood, which amounts to 2.7 times their body weight 13 . In severe infestation cases, mites can ingest as much as 6% of the total blood volume of a hen within a day 9 . Under optimal conditions (30 °C temperature and 70-85% relative humidity) the life cycle can be completed in as little as six days 14 . As a result, population densities can increase rapidly, doubling in less than 6 days 15 , even reaching 150,000-200,000 mites per hen 16 . The PRM is more prevalent in summer than in winter, and mite populations reach their peak numbers around 5 months after the start of infestation before plateauing 17 . PRM of all stages are vulnerable to low (< 30%) relative humidity 7,18 and are killed by washing the poultry houses 19 . Mites cannot withstand temperatures above 45 °C 20 and below − 20 °C 7 . Extensive farming systems provide more hiding spots for the mites and make acaricide application difficult 21 . In addition, alternative systems and backyard farms exhibit higher PRM prevalence rates 22 . Several pathogenic microorganisms have been isolated from the PRM including bacteria such as Escherichia coli and Pasteurella multocida 4 . For some of them, transmission and vector competence has also been demonstrated,, as is the case for Influenza type A virus 23 and Salmonella enterica subsp. enterica serovar Enteritidis 24,25 . Some zoonotic pathogens are a major concern for food microbiology 4 , connecting many different disciplines of biological sciences such as parasitology, bacteriology and public health safety. For instance, the zoonotic bacterium Listeria monocytogenes is an emerging food-borne pathogen 26 with reported listeriosis human cases 27 and outbreaks 28 usually attributed to contaminated poultry products instead of direct infections from infected chickens 29 . Contamination of poultry products (raw meat and eggs) might be caused directly by the hens or their environment 30,31 . Transmission can also occur through ingestion of contaminated water or airborne through contaminated soil and dust 29 . Clinical Listeria infections both in humans and in chickens are treated with antibiotics 31 . The connection between L. monocytogenes and D. gallinae up until now has been at the very least questioned 32 . L. monocytogenes has only been isolated once in a culture from PRM infesting wild animals and not chickens, more than 50 years ago with the original study presented in Russian and inaccessible to most researchers. Furthermore, no molecular tools were employed to confirm the pathogen's identity 33 . Herein, we aim to report the first molecular detection of Listeria spp. in PRM. This finding is part of a larger study investigating the haplotypes of PRM and the presence of specific pathogens, including Escherichia coli, Salmonella spp. and Listeria spp., carried by PRM in 50 different backyard poultry farms in Greece. The current work could shed light on possible transmission routes of L. monocytogenes to chickens and consequently to humans through poultry products.

Methods
Backyard chicken farm history and sampling. The backyard poultry farm was located in Central Macedonia, Northern Greece and employed a free-range system. Sample collection was performed in October 2021. Prior to sampling, the farmer gave permission to take samples and filled in a questionnaire providing all appropriate information regarding farming practices. Based on the answers, the owner had more than 30 years of experience as a poultry farmer. Thirty-five hens of different ages were kept on the poultry farm, with most of them being 52 weeks old and belonging to the Lohmann brown breed. Even though PRM had been infesting the farm for many years, the owner believed that PRM did not affect hen health and that egg production was on the expected level. However, the chickens only produced eight eggs per day, despite their age. All hens were vaccinated with commercial vaccines against Salmonella spp. and Marek's disease virus. Mites were visible on the walls (flat surfaces), but the eggs did not have any blood spots (from crushed mites). Moreover, the owner complained of feeling PRM bites on the skin and itching.
The hen house was primarily made of wood with some bricks. On the outside, trees with other birds' nests, such as swallows and pigeons, surrounded it. Chickens roamed freely in the farm's backyard and sometimes fed on the owner's home-grown vegetables. The owner did not use any egg cartons but instead collected the eggs in a bucket. Chickens did not receive any treatment for PRM, such as fluralaner, deltamethrin, diatomaceous earth, desiccant dust, or other formulations. Only cold water was used once every 3 months to wash the hen house, but with no visible effect on PRM populations. Furthermore, no monitoring devices such as cardboards were employed to assess fluctuations in PRM numbers. In order to catch the PRM, ten specially designed cardboard traps, the AviVet Red Mite Trap™, (Avivet, adVee Dierenartsen, Heeswijk Dinther, The Netherlands) were used 34 that were placed inside various cracks, perches, and nests, where mites usually hide during the day 1 . The traps covered the entirety of the hen house and were left for one week before being collected again to maximise the number of PRM caught. Consequently, traps were placed inside plastic sealed bags and transported to the School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki for further examination.

Mite identification.
A few drops of lactophenol were used to soften and clarify the mites before examining them at 100 × and 400 × magnification under an optical microscope (Olympus, CX21 Microscope). The genomic DNA of three separate mites was extracted using a commercial kit (QIAamp DNA mini kit Extraction Kit, Qiagen, Hilden, Germany) as previously described 35 . The synthetic oligonucleotide primers COI1Fyuw114 and COI1Ryuw114 35 were used in this study to amplify a partial mitochondrial Cytochrome C Oxidase subunit I PCR assay and primers for L. monocytogenes. The synthetic oligonucleotide primers (working solution 10 μM) used in this study and the size of the amplified selected fragment are listed in Table 1 36 . This primer pair amplifies a species-specific PCR product in L. monocytogenes, whereas no product is amplified in other Listeria spp. or other bacterial genera) and therefore can reliably distinguish the presence of L. monocytogenes 37  This research was carried out under the approval from the Ethics Committee of the Aristotle University of Thessaloniki (639/13-07-2020). All experiments were performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all the participants to participate in this study. Also, the farmer gave permission to publish any relevant information arising from the study.
Ethics approval and consent to participate. There was no interaction with the chickens or harm caused to them. This research was carried out under the approval from the Ethics Committee of the Aristotle University of Thessaloniki (639/13-07-2020).

Mite identification results.
In the Laboratory of Parasitology and Parasitic Diseases, mites were identified as D. gallinae based on morphological criteria 38 . The resulting PCR products from the 3 separate mites were 681 bps long based on gel electrophoresis and transillumination. DNA sequencing was successful in all 3 individual mite samples, and all 3 mites had one identical haplotype that was 520-bps long (GenBank accession number: ON597616). The haplotype identified in the current study was 99.81% similar to other PRM haplotypes previously identified in Japan 39 , further confirming that mites belonged to the species D. gallinae.

Bacterial cultures and PCR for L. monocytogenes. Microbiological cultures and confirmation tests
were only positive for L. monocytogenes (Fig. 1) and negative for E. coli and Salmonella spp. DNA was successfully extracted from TSYE agar and amplified using PCR. The resulting PCR product was 520 bps long based on gel electrophoresis and transillumination (Fig. 2), further confirming the presence of L. monocytogenes. Table 1. Nucleotide sequences of primer sets used in this study.  31 and survive in high moisture environments for many years 29 . L. monocytogenes is the primary aetiologic agent of listeriosis, an infectious disease affecting humans and birds, among other animals 26,31 .

Gene target Primer sequence (5'-3') Product size (bps) Protein encoded by the target gene
Although most bird infections are subclinical and adult chickens rarely display clinical signs, listeriosis outbreaks have been reported in backyard poultry farms 41 . Young chicks are typically more susceptible 29 , and clinical signs include diarrhoea, encephalitis, septicaemia, lower egg production 41 , and mortality 29 . Like the sampled farm in the current study, chickens living in humid and low-temperature environments with wet litter have a higher risk of infection 29 . Other predisposing risk factors that can increase environmental contamination with L. monocytogenes at the farm level include lack of sanitary measures, no vermin control programs, use of nipples without  www.nature.com/scientificreports/ cups as a watering system, presence of other animals at the farm, and inadequate disposal of faeces 42 . Noteworthy, the current backyard farm sampled had all these risk factors.
The bacterium consists of four distinct lineages 40 and, until now, 14 serotypes have been identified 43 . Human listeriosis cases can be primarily attributed to lineage I and secondarily to lineage II, with the latter mainly being isolated in food and food production facilities 40 . Listeriolysin O (LLO) is a cholesterol-dependent cytotoxin found in L. monocytogenes and is encoded by the hly gene 44 . The LLO peptide significantly increases the strain's virulence 45 and is linked with human listeriosis outbreaks 44,46 , although other genes have also been reported to affect virulence 31 . LLO-positive strains are 5-logs more virulent and spread more quickly than LLO-negative strains 46 . PCR is a fast and sensitive technique that can amplify the hly gene to confirm the presence of L. monocytogenes from selective growth media 29 , as carried out in the present study. Determination of the infecting lineage and serotype is vital in human infections in the One Health approach 47 since listeriosis is an emerging food-borne zoonotic disease 29 . Virtually all L. monocytogenes infections are food-borne 40 . Chickens with listeriosis can infect humans, and the disease can result in the patient's death in just two days 29 . Humans can also exhibit cutaneous lesions after direct contact with infected chickens or contaminated soil 29 . Contamination of poultry products, mainly chicken carcasses, is caused by poor hygiene measures and unsafe handling practices, i.e., not washing hands or cutting boards and not separating raw and cooked meat 27,48 . In our study, the specific L. monocytogenes strain was positive for the LLO protein, based on the successfully amplified hly gene.
D. gallinae in the specific poultry farm probably acquired L. monocytogenes when moving inside the poultry house 49 . Chickens can serve as natural reservoirs for the specific pathogen 50 and excrete L. monocytogenes with their faeces 51 and other secretions 29 . The farm's environment, such as dust 52 , litter 53 , soil 31 , grass 54 , water, feed 55 , decomposing vegetation 41 , nests, walls, floors, faeces, and other matrices can be contaminated with L. monocytogenes 29,30 . Transmission occurs when chickens ingest these contaminated sources, when their wounds get contaminated or when they inhale the pathogen 29 . L. monocytogenes is common in poultry in Greece and, in the past, has been isolated from 38% of poultry samples in a slaughterhouse 56 . Since mites were not washed before performing the bacteriological culture, we cannot distinguish if L. monocytogenes was harboured inside the PRM or just mechanically carried outside on its cuticle. In our study, the mites were processed according to published methodology (without washing them) [57][58][59] , though in some other studies mites were washed for example with 4% paraformaldehyde 60,61 . Mites were internally infected or externally contaminated (i.e., in their dorsal shield, genitoventral shield, legs, and chelicerae) 38 from apparently healthy chickens or other environmental sources. As demonstrated in previous studies, not washing the PRMs before processing provides more information and all-round knowledge on the vectorial potential of D. gallinae 59 . According to our results, the PRM can be added to the list of L. monocytogenes vectors, alongside carriers such as rodents, insects, and flies that can disseminate the pathogen on a farm 29 . D. gallinae can actively or passively move between facilities (i.e., poultry houses and slaughterhouses) using humans as transport hosts (hair, shoes, clothes) or through tools and equipment (egg containers, pallets, crates, and brooms, among others) 19 . In this backyard farm, chickens were mainly kept for eggs, but occasionally, some were slaughtered for their meat. The farm also had no sanitary measures in effect. Therefore, Listeria-carrying D. gallinae could suck blood from chickens and infect their wounds or get eaten by them, which represent the two most common pathogen transmission routes from PRM to hens 62,63 . Moreover, the farm had a heavy PRM infestation, as evident by the mite populations caught in the traps and mites walking on equipment that could significantly enhance the likelihood of mechanical transmission 64 and help spread L. monocytogenes. Contamination of poultry products was a real possibility that could threaten public health 65 . Suggestions were made to the farmer regarding disinfection of the farm for L. monocytogenes and treatment of hens with a licensed product for PRM.
Future research should focus on elucidating possible transmission routes of Listeria spp. Between mites and hens and detecting the same genotype in mites and meat after slaughter. That should be done on much more numerous samples (different timepoints and farms) to assess the PRM vectorial role for the specific pathogen. Nevertheless, the current work constitutes a preliminary study that helps to solidify the broad spectrum of D. gallinae as a vector of different pathogens.

Conclusions
The current work describes the first molecular isolation of L. monocytogenes from PRM, confirming that D. gallinae can carry this food-borne pathogen, which has only been questionably isolated once in the past. Control measures are required to reduce PRM populations in chicken farms, and farmers should additionally apply hygiene and sanitisation practices to minimise any possible contamination risk of poultry products with L. monocytogenes. Since antibiotics are used to treat listeriosis in hens and humans, combating D. gallinae in poultry houses could prevent transmission of L. monocytogenes, and reduce the need for antibiotics while protecting public health. The finding of L. monocytogenes further expands the vectorial role of D. gallinae highlighting how the PRM could impact other sectors, outside the sampled farm, such as food production facilities threatening public health safety. Overall, the PRM may severely affect hen health both through its haematophagous action and its ability to transmit pathogens.

Data availability
The nucleotide sequence data of the 520-bps long identical haplotype identified in all 3 individual mite samples has been deposited in GenBank (GenBank Accession Number: ON597616). www.nature.com/scientificreports/